Extracellular vesicle-embedded materials

Extracellular vesicles (EVs) are small membrane-bound vesicles released by cells. EVs are emerging as a promising class of therapeutic entity that could be adapted in formulation due to their lack of immunogenicity and targeting capabilities. EVs have been shown to have similar regenerative and therapeutic effects to their parental cells and also have potential in disease diagnosis. To improve the therapeutic potential of EVs, researchers have developed various strategies for modifying them, including genetic engineering and chemical modifications which have been examined to confer target specificity and prevent rapid clearance after systematic injection. Formulation efforts have focused on utilising hydrogel and nano-formulation strategies to increase the persistence of EV localisation in a specific tissue or organ. Researchers have also used biomaterials or bioscaffolds to deliver EVs directly to disease sites and prolong EV release and exposure. This review provides an in-depth examination of the material design of EV delivery systems, highlighting the impact of the material properties on the molecular interactions and the maintenance of EV stability and function. The various characteristics of materials designed to regulate the stability, release rate and biodistribution of EVs are described. Other aspects of material design, including modification methods to improve the targeting of EVs, are also discussed. This review aims to offer an understanding of the strategies for designing EV delivery systems, and how they can be formulated to make the transition from laboratory research to clinical use.


Introduction
Extracellular vesicles (EVs) have in recent years gained considerable attention across a wide range of interdisciplinary fields including biomaterials, bioengineering, and biomedicine.EVs are small membranebound particles actively released by various cell types into biological fluids, and play a crucial role in intercellular communication [1].Following release from donor cells, EVs may interact with receptor cells through various mechanisms, primarily classified as EV endocytosis, fusion with the cell membrane, and kiss-and-run interactions without subsequent internalisation [2].In all cases, transmembrane proteins present on the EV surface may be involved in mediating ligand-receptor interactions with the cell membrane.In the first two mechanisms, the cargo of proteins, lipids, and RNA within EVs is transferred to the recipient cell, triggering a response [3].This inherent capability of EVs to serve as natural carriers for intercellular cargo transfer has led to investigations into their potential as therapeutics.
A number of studies have revealed that EVs possess similar functions to their parent cells, making them a promising alternative to cell therapies.EV treatment can also mitigate the risk of tumor formation associated with immune rejection and cell transplantation, which is often a concern in cell therapy [4].On the other hand, EVs possess inherent advantageous characteristics including protecting their bioactive cargos from degradation in vivo, and the ability to efficiently deliver these cargos to specific targets with minimal or no immunogenicity.As a result, EVs are considered more potent candidates for therapeutic delivery than artificial nanocarriers [5].Despite the proven medical potential of EVs at the laboratory level, there are significant technical challenges in their clinical application.These challenges primarily arise from the lack of standardised protocols for EV isolation, characterisation of heterogeneous EV populations, and challenges with large-scale production of EVs, particularly EVs that remain stable in terms of their physicochemical and biological properties after isolation and formulation into an appropriate form for clinical use [6].Advancements have been made in scaling up the production of EVs to meet demand, but there remains much to be done [7].In addition, when administered systemically, EVs tend to undergo rapid clearance by the liver and kidney, resulting in insufficient concentrations in tissues and the circulatory system [8].Local injections of EVs also face limitations due to their suboptimal retention rates.To optimise the clinical utility of EVs and expand their applications, formulation strategies can be explored.Two approaches have been explored to enhance the efficiency of EVs.To begin with, various biomaterials and bioscaffolds have been developed to sustain the release of EVs, such as hydrogels, nanofibers, polymeric and inorganic scaffolds.These carriers effectively retain EVs in local tissues for extended periods while preserving their bioactivity [9].Additionally, efforts are being made to improve EV targeting by engineering vesicles, via both genetic and chemical engineering [10].
To assess and maintain the stability and functionality of EVs before and after formulation is another crucial aspect to consider in ensuring their bioactivity.The Minimal Information for Studies of EVs 2018 (MISEV2018) guidelines recommended several characterisation steps to evaluate the quality of EVs.These assessments involve evaluating factors such as vesicle integrity, protein quantification, surface markers, and functional properties of EVs [11].However, it is worth noting that comprehensive discussions on the stability of EVs before and after formulation are not always extensively covered in all research papers.It is crucial for researchers to consider and report on the stability of EVs throughout the formulation process, as it directly impacts their therapeutic potential.By ensuring the bioactivity and stability of EVs, researchers can enhance the translation and clinical applicability of EVbased therapies.
In this review, after briefly describing EV biology and their intrinsic therapeutic potential, we summarise the recent advancements in EV formulations, encompassing EV engineering and the combination of EVs with biomaterials.We will also examine the advantages of formulating EVs with a specific emphasis on enhanced stability and therapeutic effects.By elucidating the benefits of EV formulations, we aim to provide insights into their potential for clinical translation and therapeutic applications.

Discovery of EVs
Like many scientific discoveries, the discovery of EVs was accidental.Experiments that clearly identify EVs as a biological entity with enzymatic and functional potential began in 1978 [12].Prior to this, there were numerous studies that hinted at the existence of EVs.The origins of EV research started with Chargaff's coagulation study in 1945 [13].He had centrifuged blood at high speed to isolate clotting factors from cells, and observed that the high-speed sediments can significantly shorten the clotting time when added to supernatant plasma.Peter Wolf later referred to these sediments from platelet-free plasma as "platelet dust" [14].These sediments were subsequently explored by Crawford, who published images of the vesicles and named them as 'microparticles'.Crawford also suggested that these microparticles contain lipids and can carry ATP and contractile proteins [15].
Later, Nunez discovered the presence of multivesicular bodies (MVBs) by electron microscopy (EM) and suggested that the fusion of the outer or limiting membrane of the MVB with the apical plasma membrane may result in the release of internal vesicles into the luminal space (Fig. 1) [16].In 1983, Cliff Harding captured EM images of these internal vesicles as they were released upon fusion of MVBs with the plasma membrane.He further speculated a possible intracellular sorting and transport pathway, currently known as the exosome secretion pathway [17].The term "extracellular vesicle" was first reported in 1971 [18].Trams and colleagues [19] introduced the term "exosome" in 1981 to describe EVs shed from the surface of the cell, but the term was later redefined by Rose Johnstone in 1987 to specifically refer to vesicles released from the internal cavity of MVBs following fusion with the plasma membrane [20].This definition of exosomes became widely accepted and is still used today.A seminal paper published in 1996 by Raposo and colleagues [21] presented evidence for vesicles derived from the endocytic compartments of B lymphocytes, which were capable of presenting major histocompatibility complex class II (MHC II) molecules and inducing an immune response.This discovery challenged the previous understanding of EVs as simply cellular waste and highlighted their potential roles in intercellular communication and immune regulation.This paper and subsequent research in the field led to a paradigm shift in the understanding of the role of EVs in biology and medicine, and sparked significant interest in the potential therapeutic applications of EVs.

Nomenclature
The size, cargos and membrane compositions of EVs are highly heterogeneous and depend on cell origins, cell states, and environmental conditions [22].It is difficult to classify them accurately because EVs released by various biogenesis routes may carry common markers, and no specific markers have been identified so far to distinguish EV subtypes [23].Furthermore, different EV subtypes often have overlapping biophysical properties including size, density and membrane properties, and therefore cannot be completely separated by currently available methods [24].EV is accepted by the International Society of Extracellular Vesicles (ISEV) as a generic term for particles with lipid bilayers that are naturally released by cells and lack functional nuclei to replicate.To further distinguish them, the MISEV2018 protocol suggests researchers use nomenclature that reflects the physical characteristics of EVs, such as size (<100 nm or <200 nm for small EVs, >200 nm for medium or large EVs), density, specific biochemical compositions (e.g., CD63 + EVs), or origin (e.g., podocyte EVs) [11].Thus, in this review, the term "EV" is used following the MISEV2018 guidance, when there is a lack of EV categorisation or unclear evidence of the type of EV employed for a particular study.

Biogenesis of EVs
EVs are broadly divided into three subtypes including exosomes, microvesicles (MVs), and apoptotic bodies [23].Exosomes are small EVs with a mean diameter of 30 to 200 nm, which originate from endosomal membranes, and have a specific density of 1.13-1.19g/ mL [25].The biogenesis of EVs is described in Fig. 2. Exosomes are generally generated through the endosomal pathway.During this process, the plasma membrane first invaginates inward to form early endosomes, which can mature into late endosomes.The membranes of some late endosomes then bud inward to generate intraluminal vesicles (ILVs).These ILV- enriched endosomes are also named MVBs [22].ILVs carried by MVBs are transported to the plasma membrane through the cytoskeleton, where they are released into the extracellular space through exocytosis.ILVs released into the extracellular space are called exosomes [22].Some MVBs carry ILVs to the plasma membrane, while other MVBs are degraded either by directly fusing with lysosomes, or by fusing with autophagosomes and then lysosomes [26].MVs, also termed ectosomes, are large EVs with a more heterogeneous size of 50-1000 nm.They are formed and released by outward budding of the plasma membrane [27].Apoptotic bodies are released by blebbing of cells undergoing apoptosis.Their size tends to be larger (50 to 5000 nm) [28], closer to the upper limit of the EV size range [29,30].
Additionally, some cells can generate small EVs by inward budding from intracellular plasma membrane-attached vesicles (IPMCs) [25,31].A thin neck is formed by deep invagination of the plasma membrane that connects IPMCs with the extracellular environment and allows the free passage of small molecule probes and extracellular buffer.IPMCs are complex networks of interconnected membranes that can act as reservoirs for vesicle accumulation and pulsatile release.They are indistinguishable from MVBs by conventional transmission electron microscopy [32].Vesicles derived from IPMCs are similar to a subpopulation of exosomes, which express CD81, CD9, CD53, and CD63 [25,33,34].This highlights the fact that the EV population is a mixture of various EV subtypes, and the biogenesis of EVs is a complex process that involves multiple pathways and mechanisms.
EVs are commonly described as having a single lipid bilayer, but recent studies have indicated the existence of multilayered EVs [35].The formation of these multilayered structures is thought to arise via various biogenesis pathways (Fig. 3).One hypothesised mechanism is that smaller ILVs are encapsulated in larger ILVs within the MVB, but there is not strong evidence for this.Another proposed mechanism is based on the formation of lamellar bodies, secreted multilamellar organelles found in certain epithelial cells.In this process, the formation of lamellar bodies drives the generation of multivesicular structures through the flipping and rearrangement of phospholipids within the membranes [36].Additionally, the presence of ILVs adjacent to the cell membrane may lead to the formation of multilayered EVs during membrane outgrowth.In certain cases of eukaryotes, ILVs have been found to encapsulate entire membrane-bound organelles, such as mitochondria.These organelle-containing vesicles are subsequently transported to the cell surface and shed when budding outwards [37].Multilayered EVs could also form through several rounds of cellular internalisation, structural preservation, and release via membrane outgrowth.However, the supporting evidence for this is currently inconclusive.Furthermore, gram-negative bacteria can release bilayered EVs through simultaneous outgrowth of the cytoplasmic and outer membranes [38,39].This sort of EV formation is limited to prokaryotes with two membranes, while eukaryotic membrane-bound organelles in close proximity to the cell surface may experience simultaneous budding (fragmentation) with the cell membrane.Moreover, work has identified that cell protrusions on the plasma membrane of endothelial cells can form MVs that contain MVBs.After being released from the parent cells, exosomes can be stored within and subsequently released from these transporter MVs, presenting a novel site for exosome biogenesis in endothelial cells and other potential cell types [40].

Uptake and function of EVs
EVs have been shown to facilitate intercellular communications by internalisation or attachment to the cell surface.They can deliver a variety of cargos including proteins, lipids, and nucleic acids, which can affect the behaviour and phenotype of target cells [41][42][43].EVs can also deliver signalling molecules through surface-expressed ligands [44].The internalisation of EVs by recipient cells can occur through multiple mechanisms, including macropinocytosis, phagocytosis, clathrindependent endocytosis, and clathrin-independent endocytosis [45].In addition, EV uptake can also be mediated by directly fusion with the plasma membranes of a target cell [46,47].
Due to the broad heterogeneity of EVs, their contents can vary significantly.This heterogeneity is expected due to their limited carrying capacity and the various forces that drive differential distribution of biomolecules, including lipids, carbohydrates, nucleic acids and proteins, along the spectrum of endosome and plasma membranes.The multiple biogenesis pathways necessarily result in distinct compositions that are determined by the local concentration of biomolecules in the vicinity where the EVs are generated [48].Additionally, differential gene expression is another factor that contributes to the heterogeneity of EVs.For instance, EVs released by antigen-presenting cells contain high levels of MHC class II proteins and costimulatory proteins, while those released from other cell types lack these proteins [25].Environmental factors such as diet, circadian rhythms, hormones, physical activity, infections, and cell cycle stage can also drive EV heterogeneities by inducing changes in gene expression [48].This can lead to differential expression of cell surface receptors on recipient cells and can result in either survival or apoptosis [26].

Different sources of EVs and their therapeutic potential
Over the years, extensive research and clinical trials have focused on cell-based therapies, which aim to replace dysfunctional or deceased cells with functional ones, and modify the microenvironment of damaged tissue to prevent further harm and stimulate repair processes [49,50].However, increasing evidence suggests that the therapeutic effects of transplanted cells are mediated by their released components, particularly EVs, which have been shown to possess comparable or even superior therapeutic properties compared to the parent cells [51,52].As a result, EV therapy has emerged as a viable alternative to cell therapy, offering the advantage of not eliciting an immune response and thereby eliminating the need for immunosuppressive treatments [53].While the cell-free nature of EVs offers advantages in preventing cell-based infections during transport and storage [54], it is crucial to acknowledge that viruses and mycoplasma, which share size similarities with EVs, may inadvertently be included or enriched during the isolation process.Thus, rigorous quality control measures should be implemented to minimise the risk of contamination with these infectious agents.

Therapeutic effects of EVs from mesenchymal stem cells (MSCs)
MSC-derived EVs possess similar properties to MSCs, offering immunomodulation, tissue regeneration, wound healing and anti-tumor effects [55].In a mouse model of breast cancer, MSC-derived EVs enriched with the anti-angiogenic molecule miR-16 significantly attenuated the expression of vascular endothelial growth factor (VEGF), thereby inhibiting angiogenesis in breast cancer cells [56].MSC-derived EVs have also been explored in treating neurodegeneration-related disorders (due to their neuroprotective effects).Intercellular adhesion molecule 1 (ICAM1) can promote angiogenesis in human brain microvascular endothelial cells (HBMECs) through activation of the SMAD3 and P38MAPK signaling pathways.In a mouse model of Parkinson's disease (PD), MSC-derived EVs could promote angiogenesis in HBMECs by increasing the expression of ICAM1, which in turn contribute to PD recovery [57].In addition, MSC-derived EVs have been indicated to improve cardiac function and reduce infarct size after ischemiareperfusion (I/R) injury in mice.This study found that these EVs can also promote the survival of cardiomyocytes, and contribute to the recovery of cardiac function [58].Furthermore, studies have demonstrated that EVs carrying microRNAs (miRNAs) can promote the polarisation of macrophages towards the anti-inflammatory M2 phenotype, which is beneficial for treating inflammatory diseases.In one experiment, interleukin-1β (IL-1β) pretreated human umbilical cord-derived MSCs were found to release EVs with upregulated miR-146a, an anti-inflammatory miRNA that was selectively encapsulated in EVs.This led to the effective induction of M2 polarisation via EVs, which ultimately ameliorated the symptoms of sepsis [59].Macrophage polarisation to the M2 phenotype can also enhance wound healing.Studies have shown that miR-223 in bone marrow MSC-derived EVs can target the protein Pknox1, leading to the suppression of proinflammatory macrophages and the promotion of anti-inflammatory macrophages, which in turn accelerates wound healing [60].

Therapeutic effects of EVs from dendritic cells (DCs)
DC-derived EVs can carry functionally active molecules on their surfaces that participate in the formation of immunological synapses.These molecules include MHC class I and II, as well as adhesion molecules and co-stimulatory markers such as CD40, CD80, and CD86.These molecules are crucial in the induction of anti-tumor T-cell immune responses, activating and recruiting T-cells to target and destroy cancer cells [61].Given the enormous potential benefits of DC-derived EVs as an immunotherapy, they have been developed as a cell-free cancer vaccine for clinical use [62].A Phase II clinical trial utilising EVs derived from DCs on patients with end-stage non-small-cell lung cancer (NSCLC) demonstrated significant activation of natural killer cells, but no specific T-cell responses against tumor cells were observed [63].The finding indicates that immunotherapeutic methods based on DC membrane vesicles hold significant promise for use as an efficient anti-tumor treatment.

Therapeutic effects of EVs from macrophages
Macrophage-derived EVs are beneficial in cancer therapy, immunomodulation, infectious defence and tissue repair, with the precise application depending on their phenotypes and the molecules they carry [64].Research has revealed intriguingly opposite effects of EVs obtained from macrophages with different activation states.EVs produced by proinflammatory macrophages stimulate the production of Th1-promoting cytokines including IL-12 and IFN-γ by both macrophages and DC cell lines.When injected in combination with a peptide vaccine in vivo, they elicit a stronger antigen-specific cytotoxic T cell response.On the other hand, EVs derived from anti-inflammatory macrophages boost the production of anti-inflammatory cytokines IL-4 and IL-10 in macrophages and DCs [65].Additionally, these EVs carry a large number of costimulatory molecules on their surface, such as CD47, that allow them to escape immunological surveillance [66].Macrophage-derived EVs also have functions in repairing injured tissues.For instance, EVs from M2like macrophages carry miR-148a to the site of myocardial injury, and then secrete anti-inflammatory factors as well as suppressing the accumulation of excess calcium ions within the cells of the heart muscle, thereby alleviating I/R injury [67].

Therapeutic effects of EVs from tumor cells
Tumor cell-derived EVs, particularly autologous ones, are thought to carry DNA fragments, tumor antigen repertoires and costimulatory molecules comparable to their parental cells.By generating a robust T cell-dependent anti-tumor immune response, they show therapeutic effects in animal models of colon cancer, melanoma and hepatocellular carcinoma [68].By utilising intrinsic homo-adhesive properties mediated by membrane surface antigens, EVs from tumor cells have better targeting capabilities compared to EVs from non-tumor cells [69].However, it should be noted that these EVs can also have effects including the promotion of cell proliferation, inducing angiogenesis, and evading immune surveillance: these attributes can cause an acceleration of cancer progression [70].For instance, epithelial-mesenchymal transition (EMT) is a critical process in cancer cell metastasis, in which epithelial cells lose their cell-cell adhesion properties and acquire a mesenchymal phenotype, which enables them to migrate and invade surrounding tissues.EVs containing integrin-α2 subunit (ITAG2) can activate EMT in prostate cancer cells, leading to increased metastasis [71].Therefore, the safety and effectiveness of such EVs as therapeutic agents requires more investigation.

Clinical trials of EV therapies
As a consequence, EVs have attracted significant research interest.They have been explored for stem cell replacement or combination therapy with cells.According to a search on the PubMed database (30-05-2023), more than 90 % of publications published before 2023 and using the keywords "exosome" or "extracellular vesicle" were published within the last decade.Pre-clinical studies, particularly those involving stem cell-derived EV therapies, have shown encouraging results in preclinical studies including for cancer and tissue regeneration, which has resulted in several ongoing clinical trials (Table 1).The majority of these trials have used MSC-derived EVs.Other stem cells (e.g.adipose derived stem cells), immune cells (e.g.T cells, monocyte-derived DCs), and even plant cells, have also been employed to produce EVs.In addition, the HEK293 cell line has been used to produce therapeutic EVs.

EV isolation and characterisation methods
EVs can be obtained from cell culture supernatant or body fluids [74].There are several common isolation techniques available, including differential ultracentrifugation (DUC), density gradient centrifugation (DGC), size exclusion chromatography (SEC), ultrafiltration (UF), immunoaffinity capture, and polymer-based precipitation.More recently, microfluidic techniques and aptamer affinity-based methods have also been developed [75].The advantages and disadvantages of each method are summarised in Table 2.The appropriate method should be selected based on the sample source and downstream applications.DUC is often considered as the "gold standard" for EV isolation, but it can lead to the retention of proteins and lipoproteins in the EV pellets [76].Consequently, utilising a combination of separation techniques is more advantageous than relying on a single method in terms of both the resultant EV purity and yield.For instance, when SEC is employed in conjunction with low-speed centrifugation, it offers the highest level of EV purity while ensuring satisfactory EV yields [77].
Careful selection of appropriate isolation techniques can improve the quality and validity of EV samples by reducing the presence of contaminants and artifacts that may affect the accuracy and reliability of the results.However, the transformation of EVs from academia to the clinic requires compliance with large-scale Good Manufacturing Practices (GMP).Current scale-up methods are described in two recent reviews [74,86].Scaled processes require standardisation of EV enrichment, purification and quality determination.These processes can be applied to enrich EVs from various types of biofluids, such as cell culture media, plasma, serum, and urine.In the case of EVs produced from cell culture media, both static systems such as flasks and dynamic systems such as bioreactors are applied to collect conditioned media in the upstream cell Y. Ma et al. culture process.Bioreactors in particular can create a dynamic and continuous environment for cell culture, and the use of hollow fibres with molecular weight cut-off membranes allows for the acquisition of concentrated conditioned media, which is beneficial for the GMP process and downstream purification [87].Tangential flow filtration (TFF) has emerged as an alternative to DUC for purification, as it requires less time and labor for large-scale production.EVs purified by TFF have been found to exhibit higher immunomodulatory potency compared to those obtained from DUC. TFF can also avoid aggregation or destruction of EVs due to centrifugal forces [88].Another advantage of TFF is the controllable shear rate and flow rate, which prevents cake formation and subsequent vesicle fragmentation.This is in contrast to conventional UF where larger particles can often clog the membrane pores, leading to compromised separation based on the initial pore size [89].
The downstream characterisation process is critical to evaluate the quality of EVs.Detailed quality control parameters are summarised in Table 3, based on the MISEV2018 guidelines [11].Identification of EVs typically involves quantification of protein content and particle number, determination of purity, evaluation of protein markers, and characterisation of single vesicles in terms of morphology.

Routes of administration of EVs
EVs may have several benefits over other manufactured nanoparticles.They are naturally non-immunogenic when used autologously (i.e. when they are derived from the patient's own cells), while evidence regarding the immunogenicity of allogeneic EVs is currently limited [90].Plasma transfusions, which involve the transfer of trillions of allogeneic EVs, have been performed in clinical settings for many years without major immunogenic reactions.However, further research is needed to fully understand the potential immunogenicity of allogeneic EVs.Additionally, selected membrane proteins expressed on the surface of EVs, such as integrins and tetraspanins, can fuse with specific cells, allowing them to exhibit natural targeting [91].Whether they can be selectively retained at the target site or kept away from certain cells is critical to the effectiveness of EV therapy [92].While EVs have natural  targeting capabilities, these are not always sufficient for them to be recruited to the specific target site.Therefore, formulating EVs can be necessary to achieve more precise targeting and delivery to the desired location.

Systemic administration of EVs
Systemic routes like intravenous injection are commonly explored, but require the application of higher doses (as compared to local routes) to compensate for systemic clearance and non-specific uptake.Such high doses can occasionally induce adverse effects [93].The majority of EVs that are injected systemically are recognised by the mononuclear phagocyte system (MPS) and rapidly cleared from the circulation [94].Most of the free EVs will accumulate in the liver, lung and spleen in vivo [8].It is important to highlight that administration of high concentrations of EVs through systemic injection in a short timeframe may not mimic the natural biodistribution pattern of endogenous EVs.To address this limitation, micro-osmotic pumps have emerged to enable the chronic intravenous administration of EVs, mimicking the gradual and continuous manner in which EVs naturally exist and circulate in the body [95].
Recently, a study by Luo and colleagues [96] revealed notable differences in the in vivo distribution pattern of endogenous EVs released from cardiac myocytes compared to injected EVs from the same source.Under physiological conditions, endogenous EVs did not accumulate in significant amounts in the liver and spleen.This observation suggests that endogenous EVs may follow a different distribution pattern compared to injected ones [97].This difference may be attributed to the requirement to use a higher concentration of exogenous EVs to ensure their detection.To visualise and track EVs in vivo, reporters such as radioisotopes, magnetic nanoparticles or fluorescent dyes are commonly employed.These reporters often require a high EV concentration to be detectable, which may potentially trigger adverse effects in vivo, making the EVs more susceptible to clearance by the MPS [97].Furthermore, EV isolation, preparation, and storage may have negative impacts on the biological function of EVs.Contamination, low recovery rates, or aggregation may be introduced during these steps.Consequently, the paracrine and autocrine effects exhibited by EVs may be compromised or even lost.Therefore, most studies have observed similar EV distribution patterns.Another study also showed that EVs exhibit different transport patterns when administered orally and intravenously.These findings together suggest the administration method can affect EV transport patterns, and thus to potentially misleading results [98].These variations in transport patterns further emphasise the importance of considering the administration route when studying EV distribution and targeting.
In addition, the low stability of EVs and their susceptibility to nonspecific uptake pose major problems for clinical applications [99].Therefore, strategies to reduce non-specific uptake are crucial for prolonging circulation duration, minimising the applied dosage, and facilitating the distribution of EVs to target cells.One way to achieve this is through surface modifications, using techniques such as genetic engineering and chemical modifications, which can confer target specificity [10].

Genetic engineering to modify the EV surface
EVs can be genetically engineered to incorporate transmembrane proteins on the surface that can be coupled to ligands or homing peptides.This is done by transfecting donor cells with plasmids or lentiviral vectors encoding the fusion proteins.The cells then release EVs with the targeting ligands on their surface [10].This allows for the EVs to be directed towards specific cells or tissue environments with improved targeting capabilities.Lysosome-associated membrane protein 2b (Lamp2b) is currently the most extensively used modification site and is abundantly expressed on the surface of EVs.Targeting peptides can fuse with the N-terminus of Lamp2b [100].For instance, rabies virus glycoprotein (RVG) peptides have been demonstrated to specifically bind to acetylcholine receptors and have been exploited to generate neuro-specific EVs for central nervous system delivery [101].Another example is to fuse iRGD peptides with Lamp2b, which enabled EVs to interact with αv integrin on the surface of breast cancer cells, and successfully deliver incorporated doxorubicin after intravenous administration [102].Other potential binding sites for targeting ligands include CD63 (Lamp3) [103] and platelet-derived growth factor receptor (PDGFR) [104].

EV modification by chemical modification
Chemically modifying proteins by covalent conjugation of drugs, polymers and other proteins has been widely examined and, in some cases, translated to the clinic.Chemical modification of EVs has been less studied than genetic engineering.Copper-catalysed azide alkyne cycloaddition (click chemistry) has been used to conjugate both small molecules and large azide-containing model proteins to the EV surface [105].For example, in an in vivo study, researchers coupled gliomatargeting arginylglycylaspartic acid (RGE) peptides on the surface of EVs via a cycloaddition reaction with sulfonyl azide.The modified EVs were able to penetrate the blood brain barrier (BBB) and accumulate in tumor sites after being administrated intravenously in a mouse model.Meanwhile, unmodified EVs were found to be concentrated in the liver and spleen [106].Biomolecules can also be conjugated covalently onto the surface of EVs.CD47 is overexpressed on tumor cells and sends a "don't eat me" signal by binding to signal regulatory protein-α (SIRPα) on macrophages.Engineered EVs carrying SIRPα variants can act as immune checkpoint blockage by antagonising the interactions between CD47 and SIRPα.This leads to significantly enhanced tumor phagocytosis, ultimately resulting in the inhibition of tumor growth in mice with tumors [107].Similarly, EVs with high levels of CD47 isolated from adipose tissue-derived MSCs have been shown to be capable of immune escape in vivo [108].Furthermore, prior injection of EVs loaded with siRNA against clathrin heavy chain could effectively block MPS endocytosis in the spleen and liver, thereby enhancing the in vivo distribution

Table 3
The minimum EV characterisation steps required for EV quality control, based on the MISEV2018 guidelines [11]. of later injected EV doses in other target organs [94].
Other modifications include polyethylene glycol (PEG) functionalisation to improve the retention time of EVs.PEG-conjugated EVs displayed retention times improved from 10 to 60 minutes in the bloodstream of mice [109].Another study also discovered that paclitaxel-loaded aminoethylanisamide-PEG modified EVs have enhanced accumulation in lung cancer cells in mice after systemic administration [110].Moreover, modification with magnetic nanoparticles (MNPs) can allow the targeting of EVs.For instance, Li and colleagues found that by culturing superparamagnetic iron oxide nanoparticles (SPIONs) with MSCs, they were able to isolate SPIONloaded EVs.After systemic injection, applying an external magnetic field could cause EVs to accumulate at the injury site, thereby promoting skin wound healing [111].This approach allows for the targeted delivery of EVs to specific sites in the body using an external magnetic field.

EVs as colloid coating materials
Apart from the modifications of EVs, EVs themselves can also be exploited as coating materials to modify particle-based systems, improve the internalisation efficiency, and sustain the release of EVs and other cargo in the particle.An example is the use of EVs to coat immuneactivating nanocomplexes.A core-shell hybrid system was developed with the shell comprising rabies virus glycoprotein (RVG) peptide modified immature dendritic cell (imDC)-derived EVs, and the core a reactive oxygen species (ROS) responsive nanocomplex loaded with hydrophilic genes and a hydrophobic small molecular drug [112].The EV shell protects the nanocomplex core, and the delivery system can efficiently cross the BBB, target neurons, and release its therapeutic payload in the high ROS environment of diseased dopaminergic neurons [112].
In addition, EVs can be used to coat microspheres.You and colleagues prepared spherical PLGA microparticles coated with polydopamine and successfully attached EVs on the microspheres by covalent linkages [113].Microfluidic devices can be used to fabricate microspheres with EVs encapsulated internally, and these microspheres can be adsorbed onto polymer scaffolds to control the release of EVs.Specifically, EVs were loaded in triblock polymer microspheres, which were subsequently attached to nanofibrous poly(l-lactic acid) (PLLA) scaffolds [114].In this system, EVs must undergo two steps to be released, with the microspheres first detached from the scaffold, followed by the hydrolysis of the microspheres to release the EVs, resulting in a more linear release profile [114].
Nanogels have also been explored to deliver EVs through injection.Nanogels are nanoscale 3D-polymer networks composed of hydrophilic polymer chains connected through physical or chemical crosslinking.Like hydrogels, their hydrophilicity allows them to hold a large amount of water, and their nanometer size is suitable for targeted delivery of bioactive molecules like EVs.Self-assembled nanogels composed of cholesterol group-modified pullulan (CHP) have been shown to form complexes with EVs through hydrophobic interactions between the cholesterol groups of CHP and the lipid domains of EVs.This hybrid system could efficiently deliver functional EVs to cells and significantly increase the EV uptake [115].Mizuta and colleagues utilised this method and developed magnetic nanogels to deliver EVs.This study mixed magnetic iron oxide nanoparticles with CHP to form a composite nanogel with magnetic sensing ability.A hybrid of EVs and magnetic nanoparticles was formed by hydrophobic and noncovalent interactions, which generated magnetically active EVs.EV-nanogel composites were then created by mixing these two components, which can be efficiently delivered and internalised by target cells when applying an external magnetic field [116].

Controlled drug delivery systems for local delivery of EVs
Local drug delivery systems allow for the delivery of high doses of drug molecules to specific sites while limiting their distribution in other tissues.Such formulations have been widely explored for conditions including cancer and tissue repair.One example is the Gliadel wafer, which has been approved by the U.S. Food and Drug Administration (FDA) and is made of a biodegradable polymer.The wafer is surgically placed in the brain for local delivery of the chemotherapeutic drug carmustine to treat malignant glioma.It can provide sustained release of the drug to the tumor site, which enables the accumulation of a high concentration of the drug directly at the tumor site while minimising systemic side effects [117].Recent FDA-approved formulations (Table 4) demonstrate the ongoing efforts to develop localised drug delivery systems for various applications, ranging from ocular diseases to diabetes.
Similarly, there are growing interests in embedding EVs in biomaterials or bioscaffolds, which provide a more localised and concentrated EV dosage distribution.The use of biomaterials as delivery vehicles can provide enhanced stability and sustained therapeutic effects from the EVs [99].Additionally, it can allow for a controlled release of the EVs in response to changes in the environment of the diseased sites [99].To evaluate and compare the suitability of different materials for embedding EVs, it is crucial to consider the pros and cons of these materials (as summarised in Table 5) based on specific therapeutic needs and research objectives.

Hydrogels
Hydrogels are three-dimensional (3D) polymer networks formed by physical non-covalent [122] and/or chemical (covalent) crosslinking of hydrophilic polymer chains in aqueous buffer [123].Hydrogels are similar in structure to the extracellular matrix (ECM) and biological tissues, which makes them highly compatible with these structures.Hydrogels can also absorb and maintain large amounts of water or physiological fluids.These properties make them useful for creating a hydrated environment in which cellular secretions such as EVs, can be encapsulated [124].
There are several chemical crosslinking methods employed to

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Subcutaneous injection
An one-week extended release formulation that encapsulates exenatide in polymeric poly(D,Llactide-co-colide) (PLGA) microspheres to maintain glycaemic control in patients with type 2 diabetes.
N209210 [121] Y. Ma et al. formulate hydrogels, including radical polymerisation, enzyme catalysis, chemical reaction of complementary groups, and application of high energy radiation [125].The crosslink density of the hydrogel can influence its properties, including swelling, solubility, porosity and mechanical properties.Natural polymers are generally considered to be more biocompatible and bioconstructive, which make them popular choices for delivering EVs [126].Alternatively, synthetic polymers have additional features such as precise control over mechanical and physical properties, as well as chemical and thermal stability.This ability to tailor their structure and mechanical qualities makes hydrogels compatible with various types of tissues, particularly soft tissues like neural and cerebral tissues [127].Using EV-hydrogel composites has been shown to significantly improve the stability and extend the release duration of EVs [128].Therefore, hydrogels are excellent candidates for encapsulating EVs for the treatment of localised diseases.

EV release through diffusion
Several physiochemical mechanisms regulate the release of EVs from a hydrogel.These mechanisms are mainly classified into diffusion, erosion, swelling and mechanical deformation [124].Diffusion is the main release mechanism of timed delivery systems, and is usually induced by thermal motion of particles in the liquid.The speed of this spontaneous diffusive mass transport is affected by factors such as the chemical potential gradient of the system, the distance EVs have to travel to exit the gel, and the mobility of the EV particles involved in the system [129].Due to the existence of molecular networks in hydrogels, the release mechanism of trapped EVs is mainly determined by the mesh size of the matrix, polymer nature, and EV-polymer interactions.
EV diffusion in hydrogels is mainly determined by the average mesh size of the gel, which is generally heterogeneous.When EVs are smaller than the mesh size, they can be released by diffusion.The initial release of EVs from hydrogels is typically fast and driven by the high initial concentration of EVs (often in the range of 10 9 to 10 12 particles/ml) within the gel.This is known as burst release.As the concentration of EVs within the gel decreases, the release rate slows down and becomes more sustained, resulting in a release profile which follows the standard first-order model.EV release can continue for hours to days, depending on the properties of the hydrogel and its rate of degradation.Eventually, the release of EVs will reach equilibrium when the concentration inside and outside the gel becomes equal.The remaining EVs will be released only when the hydrogel is fully degraded.
When the mesh size is similar to the size of the EVs, frictional resistance occurs between the EVs and the polymer chains.Due to the heterogeneous mesh size of the hydrogel, there will be some pores smaller than the EVs, which will increase the length of their transport path [130].Consequently, the combination of diffusion and grid hindrance can slow down the EV transit out of the gel.When the EVs are larger than the mesh size, they can only diffuse out of the matrix if the network breaks due to polymer breakdown or degradation, or if the mesh expands via swelling.Increasing the crosslink density or polymer concentration can form hydrogels with high porosity and small pore size, which can effectively prolong the retention time of EVs.This has been shown in an in vitro release study in which photo-crosslinked hydrogels made from higher polymer concentrations (4 % w/v alginate) had a significantly prolonged retention time of EVs compared with hydrogels made from lower concentrations (2 % w/v alginate) [131].
Based on these findings, polymers with high swelling ratio or that swell in response to environmental stimuli are potential candidates for EV delivery.Controlling the swelling of the hydrogel can manipulate the diffusion rate of entrapped EVs.Various polymers bearing acidic (e.g.alginic acid) or basic groups have been exploited to formulate pHresponsive swelling hydrogels.In one study, nanoparticles (NPs) with similar diameter to EVs were incorporated into an alginate hydrogel.The hydrogel shrinks and seals the NPs inside under acidic conditions, but under neutral conditions deprotonation of the alginate carboxylic acid groups occurs.This results in a large amount of water ingress, and hence the gel expands to release the NPs [132].Though no pHresponsive swelling hydrogel has been studied specifically for EV delivery, such materials are promising candidates for the treatment of solid tumors or wound healing applications because the pH of injury and tumor sites is different from that of healthy tissues, and EVs can be released on demand [133,134].

EV release through degradation
Modulating the mesh degradation is a further strategy to control the release of EVs entrapped in hydrogels.The degradation of the network increases the mesh size, allowing EVs to diffuse from the hydrogel [124].Degradation can occur at crosslinks or polymer backbones as a result of hydrolysis [135], enzymatic activity [136], and other factors.Commonly used hydrolysable crosslinking molecules include anhydrides, esters and amides.Ester linkages with moderate hydrolytic halflives under physiological conditions have for instance been employed to create PEG hydrogels.By adjusting the crosslinking density and the composition of the system, the in vitro release rate of EVs could be controlled over a range from 6 to 26 days.EVs released maintained their structural and functional integrity.In a mouse model of skin wound healing, a comparison between a subcutaneously injected EV solution and hydrogel EV depot was conducted to compare the biodistribution.On day 6 after administration, the EVs in the hydrogel group were concentrated at the injection site, while free EVs spread out from the injection site within 6 hours and accumulated mostly in the liver and kidneys 2 days after administration [137].
Matrix metalloproteinases (MMPs) are responsible for biodegradation of gelatin, and have been exploited to formulate enzyme-responsive hydrogels loaded with EVs [138].One example comprises gelatin methacryloyl (GleMA) hydrogels, which can be designed to have MMPsensitive degradation properties, allowing for controlled release of EVs.Tang and colleagues [139] found that this system can significantly improve the retention of EVs in heart tissue in mice over free EVs.The hydrogel can prevent EVs from washing out, resulting in over 20 times more EVs being identified and distributed around the myocardium and endocardium.EVs remained visible in the cardiac tissue after 48 hours in the GelMA group, while most free EVs were washed out within 24 hours.Degradation of hydrogels can also be triggered by an external

Table 5
The advantages and disadvantages of materials used for formulating EVs.environmental stimulus, such as changes in pH.For example, Wang and colleagues [140] developed a pH-sensitive hydrogel composed of hyaluronic acid (HA), Pluronic F127, and poly-ε-L-lysine that can degrade more rapidly under the acidic environment in the wound area.It was found in an in vitro study that at pH 5.5, a higher percentage of EVs (80 %) were released from the hydrogel compared to pH 7.5 (65 %) on day 12. EV release can also be triggered by photodegradation of hydrogels containing ortho-nitrobenzyl ester moieties under ultraviolet (UV) light [141].By finely tuning the properties of the hydrogel, such as its degradation rate and mechanical properties, it is possible to design hydrogels that release EVs in a manner that closely matches the degradation of the material.This has been demonstrated in studies using HA hydrogel scaffolds [142].

EV release through mechanical deformation
Deformation of the hydrogel network can also affect the release of encapsulated EVs.The application of external forces, such as mechanical stress, magnetic fields, and ultrasound, can cause the deformation of the hydrogels and allow the EVs to escape from the network.Mechanical deformation can also induce convection within the network, resulting in the transient release of EVs in pulses.Hydrogels containing magnetic NPs can be deformed under the action of a magnetic field, resulting in the creation of macropores and rapid deformation of the hydrogel without mechanical damage, which leads to rapid release of loaded particles [143].Also, ultrasound can transiently disrupt the hydrogel structure.The potential benefits of ultrasound include triggering the release of the contents of hydrogels, such as nanobubbles [144], liposomes [145] and NPs [146], as well as enhancing the deep penetration and uptake of drugs within tissues [147].However, it is worth noting that external forces can damage or to some extent interfere with the EVs and render them non-functional [148].To avoid irreversible mechanical damage to hydrogels, self-healing hydrogels can be used.For example, hydrogels with reversible physical crosslinks can repair their networks after mechanical damage [149].

Hydrogel EV loading
There are three strategies for formulating hydrogel-EV composites [99].One strategy is directly mixing EVs with the hydrogel precursor solution, followed by crosslinking through the addition of crosslinking agents or physical methods.This method utilises an active precursor for covalent crosslinking, resulting in hydrogels with adjustable properties, controllable mechanical properties, and degradation rates, making them attractive materials for EV encapsulation.The use of macromonomers is also beneficial for EV delivery, as these are usually derived from biocompatible polymers, which can minimise the potential harm.Nevertheless, introducing new compounds like crosslinking agents may pose a risk of damaging the EVs [150].
The second strategy is simultaneous mixing EVs with polymers and crosslinking agents, which allows the encapsulation of EVs during the formation of the network.This method is particularly suitable for in-situ gelation, where the formulation can be injected directly at the desired position and then form a gel.Hence, it is beneficial for localised delivery of EVs to fill complex voids [99].A dual-cavity syringe is required in this approach [150].
The third strategy is called the "breathing" method, also known as the swelling method, which refers to soaking a pre-formed and lyophilised hydrogel in an EV suspension and letting it swell and become loaded with EVs [151].This method avoids any influence of polymerisation conditions on the integrity of EVs, and the formed composite can be directly lyophilised, which is convenient for transportation.The breathing method also allows for a more homogeneous distribution of EVs within the hydrogel.To employ this method for the loading of EVs into a hydrogel, the gel porosity should be large enough to allow EVs to load.If the EV particle size is bigger than the pores of the hydrogel, it will be challenging to incorporate the EVs.EVs that are loosely attached to the matrices could potentially escape through the larger pores [151].
Beyond direct encapsulation of EVs, creating physical or chemical interactions between EVs and hydrogels can improve their affinity and retention in the gels (Fig. 4).The phospholipid bilayer membranes of EVs are often decorated with various types of proteins, including spanning proteins that cross the membrane and surface markers that are attached to the outer surface of the membrane.Phosphate groups on phospholipids, and amide, amine, hydroxyl, and carboxyl groups on proteins and peptidoglycans, can interact with polar groups of polymers (e.g.carbonyl or hydroxyl groups) through hydrogen bonds or van der Waals forces [152].In one study, tannic acid (TA) was used to crosslink two components, photo-cross-linkable gelatin methacrylate (GM) and polypyrrole (PPy), to synthesise GMP hydrogels.TA can interact with the amide bonds on the GM backbone, as well as the nitrogen groups on the PPy chains as shown in Fig. 4. The polyphenol groups in TA can also form reversible hydrogen bonds with the phosphate groups on the surface of EVs, thereby effectively retaining EVs in the hydrogel and resulting in sustained in vitro release over 14 days [153].
Charge-mediated attractions between EVs and biomaterials are much stronger and more effective at retaining EVs than weaker hydrogen bonds and van der Waals interactions [154].EVs are negatively charged due to the presence of anionic phosphatidylserine (PS) and the charged residues of glycocalyx on their surface.This has led to the exploration of positively charged polymers for EV incorporation [155].For instance, chitosan (CS)-containing hydrogels, which possess a relatively weak cationic charge, can provide a prolonged release of EVs in vitro over 6 days.This study further indicated in a diabetic rat model that wounds treated with the CS-EV composite closed faster than those treated with CS alone or untreated wounds [156].Similarly, Wang and colleagues [157] loaded adipose MSC-derived EVs into pH-responsive polysaccharide-based fluorinated ethylene propylene (FEP) hydrogels via electrostatic interactions, and observed extended release for up to 21 days in acidic conditions in vitro.Compared with wounds treated by free EVs, FEP hydrogel-treated animals showed faster healing.This was thought to be due to the bioactivity of the free EVs reducing with time, as a result of degradation when they are unprotected in the in vivo environment.The FEP scaffold dressing was found to protect the bioactivity of EVs and efficiently release them to stimulate angiogenesis in both in vitro and in vivo settings.
Forming chemical covalent bonds between EVs and polymers can completely immobilise EVs in the hydrogel, and they will only be released when the network is degraded or the covalent bonds are cleaved.Covalent bonds can in principle be engineered to break down over time or triggered by external environmental stimuli.In one study, an HA hydrogel was prepared by photocleavable linkers that were attached to EVs.HA was initially conjugated with cysteine to have thiol groups.Photocleavable linker-modified EVs were then crosslinked with cysteine-modified HA hydrogels by chemical conjugation.Photocleavable linkers connected EVs with the hydrogel monomers, and the gel could thus release EV upon UV light exposure.This hydrogel system maintained the stability and integrity of EVs in vivo for several days, and provided tissue regeneration activity [141].Further, the expression of adhesion molecules on the EV surface can also enhance their affinity with biomaterials.EVs can bind to fibronectin and collagen through integrins, and bind to HA through CD44 [158].Li et al. exploited this interaction by modifying HA hydrogels with laminin-derived peptides, which effectively retained EVs by interacting with integrins on the EV surface [159].This hydrogel maintained sustained release of EVs for 11 days in vitro.According to an in vivo biodistribution study, EVs released from implanted hydrogels were retained in the injection site, while EVs given through intravenous injection were randomly distributed [159].Overall, it appears that the hydrogel platform is a promising candidate for local delivery of EVs, offering protection against degradation and clearance and potentially enhancing the therapeutic effects.

Solid polymer scaffolds
In addition to soft material scaffolds such as hydrogels, there are other solid polymer scaffolds that have been studied to deliver EVs.These involve both 2D (polymer membranes or thin films) and 3D (conventional or printed scaffolds) forms.Polymeric scaffolds serve as microporous matrices that support cell adhesion and bioactivity, and can ultimately lead to tissue regeneration in the implanted area.The presence of porosity and open channels in 2D films and 3D scaffolds are beneficial to deliver bioactive molecules and build complex composite tissue constructs [160,161].Recently, 3D printing-based additive manufacturing has emerged as a promising technique for preparing more complex geometrical scaffolds, which cannot be prepared through conventional methods [162].The release kinetics of EVs from these scaffolds can be affected by various factors, including the properties of the scaffold and the manufacturing method.Therefore, it is essential to understand the manufacturing methods and properties of scaffolds in order to optimise the EV release profile.

Release kinetics of EVs from scaffolds
Depending on how the EVs are loaded in the scaffold, there are two main delivery mechanisms: 1. release of EVs from within the scaffold, and 2. release of EVs anchored to the scaffold or their internalisation by cells located adjacent to the scaffold.In the first mechanism, EVs can attach and detach from the scaffold through adsorption and desorption, which is a dynamic reversible process described in the equation below.
Where EV⋅scaffold and EV+scaffold denote EVs attached and released from the scaffolds, and k a and k d represent the adsorption and desorption coefficients respectively.The value of k a is related to the diffusion rate of EVs, and is impacted by factors including the EV concentration difference between the scaffold and the exterior environment, and the liquid shear force present in the vicinity.Meanwhile, the value of k d correlates to the rate of EV binding to the scaffold surface, and is affected by the concentration differences and the nature of the EVsurface interactions [152].
Incubating scaffolds with EV suspensions is the simplest method to formulate EV-loaded scaffolds.Highly concentrated EV suspensions create relatively high chemical potentials, which facilitate the attachment and loading of EVs in the scaffolds.Similarly, due to the difference of chemical potentials between the scaffold and the tissue, EVs are rapidly released into the external environment after the scaffold has been implanted [114,163].As a result, simply loading EVs by adsorption is unable to maintain a stable release profile and often leads to an initial burst release.It is essential to enhance the interactions between EVs and the scaffold surface to control the release.Polydopamine (PDA)-mediated surface modification is a method to retain EVs on solid scaffolds.In a study of a poly(lactic-co-glycolic acid) (PLGA) scaffold for bone tissue engineering, PDA-coated scaffolds significantly reduced the burst release of EVs from 60 % to 20 % of the total loading on day 1, and led to an increased retention time from 4 days to over 8 days [164].PDA coating of porous nanospheres also leads to increased EV absorption and achieved more extended and controlled EV release [165].

Nanofibers
The porous structure and high surface area to volume ratio of nanofiber mats can mimic the interwoven fibrous structure of the ECM.Nanofibers are also conducive to cell adhesion, proliferation, and differentiation, making them promising scaffolds in local tissue regeneration [166].Nanofibers can improve the stability and biological activity of EVs as well as giving release over a prolonged period of time.This can be achieved by adhering EVs to the scaffold surface or by incorporating them during the fabrication process [167].Electrospinning is the most widely explored technique to produce nanofibers with controllable structure and high surface area [168].Various surface modification strategies can be used to functionalise electrospun nanofibers [169].
The principles of the electrospinning technology are described in detail in recent reviews [170].Bioactive agents, including EVs, can be incorporated in nanofibers by using single-needle and/or coaxial electrospinning methods.Studies have demonstrated that embedding EVs into nanofibers can improve their stability.Németh and colleagues [167] successfully formulated EV-polyvinylpyrrolidone (PVP) nanofibers by mixing EVs with a PVP solution and then electrospinning.The EV-loaded nanofibers maintained their fibrillar structure for up to 12 weeks, independent of temperature.This can be explained by dint of the fact that PVP can be attached to the EV surface by hydrogen bonding to form a protective shell of macromolecules, thus increasing the stability of the EVs and preventing aggregation.Currently, only single-needle systems have been applied to formulate EV-entrapped nanofibers, but there have been studies of successfully embedding liposomes into the core of coaxial fibres [171].
It is also possible to attach EVs to the nanofiber surface, for instance by electrostatic adsorption.Su and colleagues conjugated PEG molecules to the surface of poly(ε-caprolactone) (PCL) fibres, giving a positive substrate for EV adsorption.Over 40 % of the EVs were still retained after one week in vitro, and the release of EVs was mainly through active uptake by cells in contact with the scaffold rather than passive release [172].PDA-coating is also effective in loading bioactive factors [173].Researchers coated PDA on the surface of electrospun fibres to adhere EVs, and found it significantly improved the loading capacity by 1.4-fold compared to regular physical adsorption or conjugation to phospholipids on the EV surface.In an in vitro release study, PDA-coated nanofibers retained nearly 90 % of their EVs over 12 days [174].

3D-bioprinted scaffolds
3D printing, also known as additive manufacturing, enables precise fabrication of scaffolds with well-defined structure and shape.EVs can either be loaded into/onto the scaffold after its fabrication or printed directly with the scaffold using a bio-ink composed of EVs, polymer, and potentially other biomaterials.Chen and colleagues [175] developed a 3D-printable bio-ink consisting of MSC-derived EVs, decellularised cartilage ECM, and gelatin methacrylate (GelMA).The bio-ink possessed a photo-crosslinking function and was printed to obtain a radially oriented 3D hydrogel scaffold, which retained EVs in vitro for 2 weeks and facilitated the migration of chondrocytes to promote cartilage regeneration.In another study, a bio-ink composed of EVs, sodium alginate and HA was printed layer by layer by extrusion technology and then crosslinked with CaCl 2 solution to form a hydrogel scaffold [176].The 3Dprinted scaffold formed structures with a uniform EV distribution.An in vitro release study exhibited an initial burst release within 24 h, followed by a more gradual and sustained release, with around 80 % of the EVs retained in the gel after a month.The system could enhance tissue repair by modulating the inflammatory response [176].However, direct attachment of EVs to 3D-printed scaffolds may result in low loadings, so surface modifications of EVs or the scaffolds are often required to improve their adhesion.Zha and colleagues [177] utilised the EV anchoring peptide CP05 to modify a 3D-printed PCL scaffold to specifically bind to CD63 on the surface of the EVs, thereby improving the loading efficiency.Another option is to use polyethyleneimine (PEI), a biocompatible polymer with a "proton sponge" effect: protonation causes the polymer to swell, resulting in enlarged pores that facilitate the release of an internal cargo.PEI has been exploited to modify the surface of 3D-printed porous PLA scaffolds and thereby improve EV adhesion.In one study, PEI-coated scaffolds effectively increased the retention of EVs with enhanced in vitro cellular internalisation.After implantation in vivo for 6 weeks, this system was found to stimulate bone regeneration in rats [178].However, it is worth noting that cationic polymers such as PEI also have potential drawbacks, including demonstrated toxicity [179].

Solid inorganic scaffolds
Due to their stiffness and biocompatibility, inorganic scaffolds are widely explored as EV delivery systems for bone tissue regeneration.Commonly used inorganic materials include metals [180], hydroxyapatite [181], tricalcium phosphate (TCP) [163], ceramics [182] and bioactive glass (BAG) [183].Clinically, metals are the most widely used, especially in bone implants and cardiovascular stents.Titanium alloys are commonly used in orthopaedics due to their good biocompatibility and corrosion resistance [184].In addition, micro/nano-textured layered titanium can also promote EV release and biogenesis, thereby promoting bone regeneration [185].
EV-modified metal systems have been shown to give enhanced bone regeneration and repair effects over conventional scaffolds, for instance in the case of porous titanium alloy scaffolds and titanium oxide nanotubes [180,186].Recently, metal-organic framework (MOF) scaffolds for delivering EVs have been proposed to have promise in bone tissue regeneration.MOFs are composed of organic ligands connecting metal ions or metal cluster nodes through coordination bonds, and have useful properties such as high surface area, adjustable porosity and biocompatibility [187].Kang and colleagues [188] used electrospinning to prepare PLGA-MOF composite scaffolds loaded with magnesium ions and gallic acid, and tethered EVs to the surface through electrostatic interactions.The scaffold showed in vitro release of EVs over up to 10 days.The slow release of magnesium ions created a high-magnesium environment that accelerated cell adhesion and growth.The study observed new bone formation in rat calvarial bone defects at 5 and 10 weeks post-implantation.At the 10-week mark, the EV-scaffold group showed greater bone formation (with the defective areas covered by collagen and new bone tissue) compared to the control group (which primarily contained fibroblasts and proliferating fibrous tissue).These results suggest that the EV scaffold has excellent osteoconductive capacity and potential to promote new in vivo bone growth [188].
Many inorganic scaffolds require high-temperature sintering to burn off a polymer template, which is not compatible with incorporating EVs into the scaffold during initial synthesis.Currently, EVs are often loaded onto inorganic scaffolds by infiltration.Liu and colleagues explored different polymer templates to manufacture a hierarchical mesoporous bioactive glass (MBG) scaffold with macro, micro and meso porosities [189].This scaffold includes polyurethane sponges forming large pores (200-500 μm), Pluronic F-127 micelles forming mesopores (7.7 nm), and lyophilised EVs packaged in micropores (0.5-2 μm).The high surface area and microporosity of this layered MBG scaffold provided an ideal environment for protecting EVs from disruption during the lyophilisation process, leading to preserved bioactivity.This feature of the scaffold makes it highly promising for use in bone repair applications [189].Being relatively biologically inert, most inorganic scaffolds are surfacemodified with coatings to enhance their interaction with EVs and ensure prolonged release.Studies indicate that layer-by-layer self-assembly technology can alternately deposit positively charged poly-L-lysine (PLL) polypeptides and negatively charged HA on β-tricalcium phosphate (β-TCP) scaffolds.This biomimetic coating improves the immobilisation of EVs on the scaffold [190].Additionally, nanocement matrix (NC) was also utilised as a carrier to deliver EVs.Qayoom and colleagues reported a calcium sulfate and nanohydroxyapatite-based NC system that enabled controlled release of bone marrow MSC-derived EVs and other bioactive molecules.The gradual dissolution of calcium sulfate facilitates the formation of pores and the release of encapsulated EVs.This slow dissolution process also provides sufficient space for cell attachment and infiltration, which promoted bone healing [182].

Conclusion and future perspectives of EV delivery system
As cell-free therapeutics, EVs possess numerous advantages due to their ability to carry a variety of biomolecules, the lack of immunogenicity and targeting capabilities.In particular, EVs derived from MSCs have shown potential in multiple medical conditions.However, the clinical application of EVs is still in its infancy and there are several challenges that need to be addressed.A lack of standardisation in the EV isolation and processes is a key reason why clinical studies fall short of their goals.Beyond this, there are other variations preventing their clinical use, including differences in purification procedures that can result in potential heterogeneity of the product EV populations.Nonetheless, the accessibility of EVs and their potential to cross physiological barriers make them highly promising for future clinical applications.Even though there have been many studies on EVs in recent years, some crucial parameters still need to be established in order to prevent offtarget effects on other organs that may lead to toxicity or tumorgenesis.These parameters include identifying the specific EV subpopulations responsible for therapeutic effects and determining the optimal mode of administration One potential solution is to formulate EVs with biomaterials or bioscaffolds, which are already known and used with cell therapy.This approach could mitigate the potential risk of undesired EV distribution in the body.Such advanced formulations can also provide sustained release of EVs and protect them from rapid clearance by the immune system.A number of pre-clinical studies have shown the enhanced stability of encapsulated or carried EVs.Additionally, the carrier biomaterials themselves are able to provide supportive or tissue-repairing properties.Various types of EV delivery systems have been described in this review, including hydrogels, nanofibers, polymer-based scaffolds, and inorganic materials.Modification of scaffolds can improve their mechanical properties, biocompatibility, and EV-binding ability.While associating EVs with scaffolds is a potential solution to limit off-target effects, there are also several challenges that need to be addressed.For example, it is necessary to investigate how the scaffolds might affect the EV cargo over extended periods of time.Additionally, it is crucial to develop suitable sterilisation, storage and transportation methods for material-EV composites and determine whether these post-fabrication processes compromise the integrity and therapeutic efficacy of the EVs.Most importantly, in vivo studies of the biodistribution of EVs are required to further evaluate the safety and efficacy of these systems.Once these challenges are addressed, advanced drug delivery systems of EVs have the potential to revolutionise therapeutic interventions and provide new opportunities for the treatment of a variety of diseases, including cancer therapy, regenerative medicine, and gene therapy.

Declaration of Competing Interest
None.

Fig. 1 .
Fig. 1.Schematic representation of the release of intraluminal vesicles (ILVs) into the luminal space by fusion between the multivesicular body (MVB) and the plasma membrane.(Drawn in Biorender)

Fig. 2 .
Fig. 2. Extracellular vesicle (EV) biogenesis pathways include: 1. the endosomal pathway, in which vesicles are formed by the inward invagination of the endosome membrane and further mature into ILV-enriched MVBs.These MVBs can either be degraded through autophagy by lysosomes or be released through exocytosis, after which they are named exosomes.2. The plasma membrane pathway, in which microvesicles (MVs) are formed by the outward budding of the plasma membrane.3. The intracellular plasma membrane-attached vesicles (IPMCs) pathway, in which small extracellular vesicles (EVs) are stored in the compartment of IPMCs and released in a pulsatile manner.4. The apoptotic bodies pathway, in which vesicles are released by blebbing of cells undergoing apoptosis.(Drawn in Biorender)

Fig. 4 .
Fig. 4. Methods to improve the affinity between EVs and hydrogels through various physical or chemical interactions.1. Creating intermolecular forces between surface proteins on EVs and polymers, such as hydrogen bonds, Van der Waals forces and hydrophobic interactions.2. Utilising electrostatic interactions between negatively charged phosphatidylserine (PS) on the EV surface and positively charged polymers.3. Establishing covalent bonds between EVs and polymers.4. Binding of adhesion molecules on the EV surface and polymers such as collagen, fibronectin and hyaluronic acid (HA) via receptor-ligand interactions.(Drawn in Biorender)

Table 1
A list of publicly disclosed clinical trials of EV therapies.Data collected from https://clinicaltrials.gov/ct2/home on 30-May-2023.

Table 2
The advantages and disadvantages of different isolation methods to produce EVs.